1.1 Field of the Invention
The present invention relates generally to the fields of molecular biology. More particularly, certain embodiments concern methods and compositions comprising DNA segments, and proteins derived from bacterial species. More particularly, it concerns novel cryET33 and cryET34 genes from Bacillus thuringiensis encoding coleopteran-toxic crystal proteins. Various methods for making and using these DNA segments, DNA segments encoding synthetically-modified Cry proteins, and native and synthetic crystal proteins are disclosed, such as, for example, the use of DNA segments as diagnostic probes and templates for protein production, and the use of proteins, fusion protein carriers and peptides in various immunological and diagnostic applications. Also disclosed are methods of making and using nucleic acid segments in the development of transgenic plant cells containing the DNA segments disclosed herein.
1.2 Description of the Related Art
1.2.1 Bacillus thuringiensis Crystal Proteins
One of the unique features of B. thuringiensis is its production of crystal proteins during sporulation which are specifically toxic to certain orders and species of insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions including B. thuringiensis strains which produce proteins having insecticidal activity against lepidopteran and dipteran insects have been commercially available and used as environmentally-acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
The mechanism of insecticidal activity of the B. thuringiensis crystal proteins has been studied extensively in the past decade. It has been shown that the crystal proteins are toxic to the insect only after ingestion of the protein by the insect. The alkaline pH and proteolytic enzymes in the insect mid-gut solubilize the proteins, thereby allowing the release of components which are toxic to the insect. These toxic components disrupt the mid-gut cells, cause the insect to cease feeding, and, eventually, bring about insect death. For this reason, B. thuringiensis has proven to be an effective and environmentally safe insecticide in dealing with various insect pests.
As noted by Höfte et al., (1989) the majority of insecticidal B. thuringiensis strains are active against insects of the order Lepidoptera, i.e., caterpillar insects. Other B. thuringiensis strains are insecticidally active against insects of the order Diptera, i.e., flies and mosquitoes, or against both lepidopteran and dipteran insects. In recent years, a few B. thuringiensis strains have been reported as producing crystal proteins that are toxic to insects of the order Coleoptera, i.e., beetles (Krieg et al., 1983; Sick et al., 1990; Lambert et al., 1992).
1.2.2 Genetics of Crystal Proteins
A number of genes encoding crystal proteins have been cloned from several strains of B. thuringiensis. The review by Höfte et al. (1989) discusses the genes and proteins that were identified in B. thuringiensis prior to 1990, and sets forth the nomenclature and classification scheme which has traditionally been applied to B. thuringiensis genes and proteins. cryI genes encode lepidopteran-toxic CryI proteins. cryII genes encode CryII proteins that are toxic to both lepidopterans and dipterans. cryIII genes encode coleopteran-toxic CryII proteins, while cryIV genes encode dipteran-toxic CryIV proteins.
Recently a new nomenclature has been proposed which systematically classifies the cry genes based upon DNA sequence homology rather than upon insect specificities. This classification scheme is shown in Table 1.
TABLE 1Revised B. thuringiensis δ-Endotoxin Gene NomenclatureaNewOldGenBank Accession #Cry1AaCryIA(a)M11250Cry1AbCryIA(b)M13898Cry1AcCryIA(c)M11068Cry1AdCryIA(d)M73250Cry1AeCryIA(e)M65252Cry1BaCryIBX06711Cry1BbET5L32020Cry1BcPEG5Z46442Cry1CaCryICX07518Cry1CbCryIC(b)M97880Cry1DaCryIDX54160Cry1DbPrtBZ22511Cry1EaCryIEX53985Cry1EbCryIE(b)M73253Cry1FaCryIFM63897Cry1FbPrtDZ22512Cry1GPrtAZ22510Cry1HPrtCZ22513Cry1HbU35780Cry1IaCryVX62821Cry1IbCryVU07642Cry1JaET4L32019Cry1JbET1U31527Cry1KU28801Cry2AaCryIIAM31738Cry2AbCryIIBM23724Cry2AcCryIICX57252Cry3ACryIIIAM22472Cry3BaCryIIIBX17123Cry3BbCryIIIB2M89794Cry3CCryIIIDX59797Cry4ACryIVAY00423Cry4BCryIVBX07423Cry5AaCryVA(a)L07025Cry5AbCryVA(b)L07026Cry5BU19725Cry6ACryVIAL07022Cry6BCryVIBL07024Cry7AaCryIIICM64478Cry7AbCryIIICbU04367Cry8ACryIIIEU04364Cry8BCryIIIGU04365Cry8CCryIIIFU04366Cry9ACryIGX58120Cry9BCryIXX75019Cry9CCryIHZ37527Cry10ACryIVCM12662Cry11ACryIVDM31737Cry11BJeg80X86902Cry12ACryVBL07027Cry13ACryVCL07023Cry14ACryVDU13955Cry15A34kDaM76442Cry16Acbm71X94146Cyt1ACytAX03182Cyt2ACytBZ14147To Be AssignedCryET33, Present InventionTo Be AssignedTo Be AssignedCryET34, Present InventionTo Be Assigned1.2.3 Identification of Crystal Proteins Toxic To Coleopteran Insects
The utility of bacterial crystal proteins as insecticides was extended when the first isolation of a coleopteran-toxic B. thuringiensis strain was reported (Kreig et al., 1983; 1984). This strain (described in U.S. Pat. No. 4,766,203, specifically incorporated herein by reference), designated B. thuringiensis var. tenebrionis, is reported to be toxic to larvae of the coleopteran insects Agelastica alni (blue alder leaf beetle) and Leptinotarsa decemlineata (Colorado potato beetle).
U.S. Pat. No. 4,766,203 (specifically incorporated herein by reference) relates to a 65-70 kilodalton (kDa) insecticidal crystal protein identified in B. thuringiensis tenebrionis (see also Berhnard, 1986). Sekar et al., (1987) report the cloning and characterization of a gene for a coleopteran-toxic crystal protein from B. thuringiensis tenebrionis. The predicted size of the polypeptide (as deduced from the gene sequence) is 73 kDa, however, the isolated protein consists primarily of a 65-kDa component. Höfte et al. (1987) also reports the DNA sequence for the cloned gene from B. thuringiensis tenebrionis, with the sequence of the gene being identical to that reported by Sekar et al. (1987).
McPherson et al. (1988) discloses a DNA sequence for the cloned insect control gene from B. thuringiensis tenebrionis; the sequence was identical to that reported by Sekar et al. (1987). E. coli cells and Pseudomonas fluorescens cells harboring the cloned gene were found to be toxic to Colorado potato beetle larvae.
Intl. Pat. Appl. Publ. No. WO 91/07481 dated May 30, 1991, describes B. thuringiensis mutants that produce high yields of the same insecticidal proteins originally made by the parent strains at lesser yields. Mutants of the coleopteran-toxic B. thuringiensis tenebrionis strain are disclosed.
A coleopteran-toxic strain, designated B. thuringiensis var. san diego, was reported by Herrnstadt et al. (1986) to produce a 64-kDa crystal protein toxic to some coleopterans, including Pyrrhalta luteola (elm leaf beetle); Anthonomus gradis (boll weevil), Leptinotarsa decemlineata (Colorado potato beetle), Osiorhynchus sulcatus (black vine weevil), Tenebrio molitor (yellow mealworm), Haltica zombacina; and Diabrotica undecimpuncata undecimpunctata (western spotted cucumber beetle).
The DNA sequence of a coleopteran toxin gene from B. thuringiensis san diego was reported by Herrnstadt et al. (1987); and was disclosed in U.S. Pat. No. 4,771,131. The sequence of the toxin gene of B. thuringiensis san diego is identical to that reported by Sekar et al. (1987) for the cloned coleopteran toxin gene of B. thuringiensis tenebrionis. Krieg et al., (1987) demonstrated that B. thuringiensis san diego was identical to B. thuringiensis tenebrionis, based on various diagnostic tests.
Another B. thuringiensis strain, EG2158, was reported by Donovan et al. (1988) and described in U.S. Pat. No. 5,024,837. EG2158 produces a 73-kDa CryC crystal protein that is insecticidal to coleopteran insects. Its DNA sequence was identical to that reported by Sekar et al. (1987) for the cloned B. thuringiensis tenebrionis toxin gene. This coleopteran toxin gene is referred to as the cryIIIA gene by Höfte et al., 1989. Two minor proteins of 30- and 29-kDa were also observed in this strain, but were not further characterized (Donovan et al., 1988).
U.S. Pat. No. 5,024,837 also describes hybrid B. thuringiensis var. kurstaki stains which showed activity against both lepidopteran and coleopteran insects. U.S. Pat. No. 4,797,279 (corresponding to EP 0221024) discloses a hybrid B. thuringiensis transformed with a plasmid from B. thuringiensis var. kurstaki containing a lepidopteran-toxic crystal protein-encoding gene and a plasmid from B. thuringiensis tenebrionis containing a coleopteran-toxic crystal protein-encoding gene. The hybrid B. thuringiensis strain produces crystal proteins characteristic of those made by both B. thuringiensis kurstaki and B. thuringiensis tenebrionis. U.S. Pat. No. 4,910,016 (corresponding to EP 0303379) discloses a B. thuringiensis isolate identified as B. thuringiensis MT 104 which has insecticidal activity against coleopterans and lepidopterans.
European Pat. Appl. Publ. No. 0318143 discloses an intact, partially-modified gene from B. thuringiensis tenebrionis and recombinant vectors comprising it able to direct expression of a protein having toxicity to coleopteran insects, and Eur. Pat. Appl. Publ. No. 0324254 discloses B. thuringiensis A30; a strain which has insecticidal activity against coleopteran insects, including Colorado potato beetle larvae, corn rootwormn larvae and boll weevils.
U.S. Pat. No. 4,999,192 (corresponding to EP 0328383) discloses B. thuringiensis PS40D1 which has insecticidal activity against Colorado potato beetle larvae. The strain was also identified via serotyping as being serovar 8a8b, morrisoni. U.S. Pat. No. 5,006,336 (corresponding to EP 0346114) described a B. thuringiensis isolate, designated PS122D3, which was serotyped as serovar 8a8b, morrisoni and which exhibited insecticidal activity against Colorado potato beetle larvae. U.S. Pat. No. 4,966,765 (corresponding to EP 0330342) discloses a B. thuringiensis strain, PS86B1 (identified via serotyping as being serovar tolworthi), which has insecticidal activity against the Colorado potato beetle.
The nucleotide sequence of a cryIIIB gene and its encoded coleopteran-toxic protein is reported by Sick et al., (1990) but the B. thuringiensis source strain is identified only via serotyping as being subspecies tolworthi. U.S. Pat. No. 4,966,155, issued Feb. 26, 1991, of Sick et al. (corresponding to EP 0337604), discloses a B. thuringiensis toxin gene obtained from the coleopteran-active B. thuringiensis 43F, and the gene sequence appears identical to the cryIIIB gene. B. thuringiensis 43F is reported as being active against Colorado potato beetle and Leptinotarsa texana. 
Eur. Pat. Appl. Publ. No. 0382990 discloses two B. thuringiensis strains, btPGS1208 and btPGS1245, which produce crystal proteins of 74- and 129-kDa, respectively, that exhibit insecticidal activity against Colorado potato beetle larvae. The DNA sequence reported for toxin gene producing the 74-kDa protein appears to be related to that of the cryIIIB gene of Sick et al (1990).
PCT Intl. Pat. Appl. Publ. No. WO 90/13651 discloses B. thuringiensis strains which contain a toxin gene encoding an 81-kDa protein that is said to be toxic to both lepidopteran and coleopteran insects. U.S. Pat. No. 5,055,293 discloses the use of B. laterosporous for corn rootworm (Diabrotica) insect control.